Mammalian tissues are dependent upon the continuous supply of oxygen and glucose needed for the energy production. This energy is used for various types of work, including the maintaining of ionic balance and biosynthesis of various cellular components. The ratio or balance, between oxygen supply and demand reflects the cells' functional capacity to perform their work. In this way, the energy balance reflects the metabolic state of the tissue. In order to assess the tissue energy balance, it is necessary to monitor the events continuously using a multiparametric system in real-time.
The integrated system of energy supply and demand can be understood by considering the various components thereof.
O2 supply: The blood carries the oxygen and other essential substances to the cells. Therefore, monitoring of blood flow rate, blood volume and blood oxygenation will reflect the supply of O2 to the tissue for the purpose of energy formation therein.
Energy production and demand: In an inner compartment of the cells, called the mitochondria, the glucose and O2 are transformed into ATP, a form of energy which can be used by the cells for various types of activities. The ATP production rate is, in normal states, regulated by rate of consumption of ATP, and is increased when cellular activity rises. In most pathological states, the limiting factor for this process is O2 availability.
The process of energy (ATP) production and consumption can be determined through monitoring of Nicotineamide adenine dinucleotide (NADH) redox state. The NADH and NAD molecules can be correlated with the process of ATP production. The concentration of the reduced form of the molecule (NADH) rises when the rate of ATP production is low, and is unable to meet the demand in the tissue or cells.
A complementary indicator of energy production, other than NADH, is the concentration of flavoproteins (Fp). Flavoprotein molecules are also linked to the production of ATP in the mitochondria. Fp concentration drops when the rate of ATP production is reduced, and is unable to meet the demand in the tissue or cells.
There is direct correlation between energy metabolism of the cellular compartment and the blood flow in the microcirculation of the same tissue. In a normal tissue, any change in the O2 demand will be compensated by a corresponding change in the blood flow to the tissue. By this mechanism, the O2 supply remains constant if there is no change in the O2 consumption. Any change in the abundance of O2 in the tissue, in other words a change in energy state, will be reflected by the NADH and Fp level.
It is important to monitor both supply and demand in order to be able to detect pathological situations in which the balance is disrupted, and one component of the system reacts abnormally with respect to the other.
The parameters used in the art for the assessment of tissue vitality include: A—Blood Flow Rate; B—Mitochondrial Redox State via the NADH level; C—Blood Volume; D—Blood Oxygenation State; E—Mitrochondrial Redox State via flavoprotein level.
A—Blood Flow Rate
The blood flow rate relates to the mean volume flow rate of the blood and is essentially equivalent to the mean velocity multiplied by the number of moving red blood cells in the tissue. This parameter may be monitored by a technique known as Laser Doppler Flowmetry, which is based on the fact that light reflected off moving red blood cells (RBC) undergoes a small shift in wavelength (Doppler shift) in proportion to the cell's velocity. Light reflected off of stationary RBC or bulk stationary tissue, on the other hand, does not undergo a Doppler shift.
By illuminating with coherent light, such as a laser, and converting the intensities of incident and reflected light to electrical signals, it is possible to estimate the blood flow from the magnitude and frequency distribution of those signals (U.S. Pat. No. 4,109,647; Stem, M. D. Nature 254, 56–58, 1975).
B—Mitochondrial Redox State or the NADH Level
The level of Nicotineamide adenine dinucleotide (NADH), the reduced form of NAD, is dependent both on the availability of oxygen and on the extent of tissue activity. Referring to FIG. 1, whilst NADH absorbs UV light at wavelengths of about 300 nm to about 400 nm and fluoresces at wavelengths of about 400 nm to about 550 nm, the NAD does not fluoresce. The NADH Level can thus be measured using Mitochondrial NADH Fluorometry. The conceptual foundations for Mitochondrial NADH Fluorometry were established in the early 50's and were published by Chance and Williams (Chance B., & Williams G. R., Journal of Biological Chemistry, 217, 383–392, 1955). They defined various metabolic states of activity and rest for in-vitro mitochondria.
An increase in the level of NADH with respect to NAD and the resulting increase in fluorescence intensity indicate that insufficient Oxygen is being supplied to the tissue. Similarly, a decrease in the level of NADH with respect to NAD and the resulting decrease in fluorescence intensity indicate an increase in tissue activity.
C—Blood Volume
The blood volume parameter refers to the concentration of the blood in the tissue. When tissue is irradiated, the intensity R of reflection of the excitation wavelength light from the tissue is informative of the blood volume. The intensity R of the reflected signal, also referred to as the total backscatter, increases dramatically as blood is eliminated from the tissue as a result of the decrease in haemoglobin concentration. Similarly, if the tissue becomes more perfused with blood, R decreases due to the increase in the haemoglobin concentration.
D—Blood Oxygenation State
The blood oxygenation state parameter refers to the relative concentration of oxyhaemoglobin to deoxy-haemoglobin in the tissue. It may be assessed by the performance of photometry measurements. The absorption spectrum of oxyhaemoglobin HbO2 is considerably different from the absorption spectrum of deoxy-haemoglobin Hb (Kramer R. S. and Pearlstein R. D., Science, 205, 693–696, 1979). The measurement of the absorption at one or more wavelengths can thus be used to assess this important parameter. Blood oximeters are based on measurement of the haemoglobin absorption changes as blood deoxygenates (Pologe J. A., Int. Anesthesiol. Clin., 25(3), 137–53, 1987). Such oximeters generally use at least two light wavelengths to probe the absorption. One known method uses one wavelength at an isosbestic point and another wavelength at a point that exhibits absorption changes due to variation in oxygenation level. Another technique uses wavelengths at both sides of an isosbestic point in order to increase measurement sensitivity. The wavelengths used in commercial pulse oximeters are typically around 660 nm in the red region of the spectrum, and between 800 to 1000 nm in near-infrared region (Pologe, 1987).
Isosbestic point as referred to herein is a wavelength at which the intensity of absorption of oxyhaemoglobin HbO2 is the same as the intensity of absorption of deoxy-haemoglobin Hb; to such isosbestic points are indicated as IPA and IPB in FIG. 10. Similarly, there is an isosbestic range marked IR in FIG. 10 where these two functions are substantially coincident. FIG. 10 is based on Anderson, R. R., Parrish, J. A. (1981) Microvasculature can be selectively damaged using dye lasers: a basic theory and experimental evidence in human skin. Lasers Surg. Med. 1, 263–276.
For monitoring the oxygenation levels of internal organs, fiber-optic blood oximeters have been developed. These fiber-optic devices irradiate the tissue with two wavelengths, and collect the reflected light. By analysis of the reflection intensities at several wavelengths the blood oxygenation is deduced. The wavelengths used in one such system were 585 nm (isosbestic point) and 577 nm (Rampil I. J., Litt L., & Mayevsky A., Journal of Clinical Monitoring, 8, 216–225, 1992). Another blood oximeter measures and analyzes the whole spectrum band 500–620 nm (Kessler M. & Frank K., Quantitative spectroscopy in tissue pp. 61–74. Verlagsgruppe GmbH, Frankfurt au Main, 1992). These devices are relatively complicated and susceptible to interference from ambient light, as well as various electronic and optic drifts. Two light sources are required, and the light sources and the detection system also incorporate optical filters that are interchangeable by mechanical means.
E—Flavoprotein Concentration
In order to determine the metabolic state of various tissues in-vivo it is also possible to monitor the fluorescence of another cellular fluorochrome, namely Flavoproteins (Fp). Referring to FIG. 12, Fp absorbs light at wavelengths of about 400 nm to about 470 nm and fluoresces at wavelengths of about 490 nm to about 580 nm. The Fp level can thus be measured using Fp Fluorometry. The conceptual foundations for Fp Fluorometry were established in the late 1960's and were published in several papers as will be referenced hereinafter. Simultaneous monitoring of NADH and Fp from the same layer or volume of tissue provides better interpretation of the changes in energy production and demand.
Chance et al.(B. Chance, N. Graham, and D. Mayar. A time sharing fluorometer for the readout of intracellular oxidation-reduction states of NADH and Flavoprotein. The Review of Scientific Instruments 42 (7):951–957, 1971) used a time-sharing fluorometer to record intracellular redox state of NADH and Fp. They showed a very clear correlation between the two chromophores to changes in O2 supply to the perfused liver. Using a time sharing fluorometer reflectometer simultaneous monitoring of NADH and Fp was performed from the surface of the rat's brain (A. Mayevsky. Brain energy metabolism of the conscious rat exposed to various physiological and pathological situations. Brain Res. 113:327–338, 1976). The kinetics of the responses to anoxia or decapitation were identical for the NADH and Fp indicating that the NADH signal comes from the same cellular compartment as the Fp—the mitochondrion.
The five tissue viability parameters described above represent various important biochemical and physiological activities of body tissues. Monitoring them can provide much information regarding the tissues' vitality. For the monitoring of different parameters to have maximum utility however, the information regarding all parameters is required to originate from substantially the same layer of tissue, and preferably the same volume of tissue, otherwise misleading results can be obtained. In general, the more parameters that are monitored from the same tissue volume or layer, the better and more accurate an understanding of the functional state of the tissue that may be obtained.
There are several techniques that relate to the simultaneous in-vivo measuring of multiple parameters in certain tissues, which can be used for the various pathological situations arising in modem medicine.
The prior art teaches a wide variety of apparatuses/devices which monitor various parameters reflecting the viability of the tissue. For example, U.S. Pat. No. 4,703,758 teaches the use of an apparatus for monitoring blood flow by using a light source to emit a beam of light, and a light detector that measures the light received. This provides the value of the intensity of the transmitted light, which inter alia depends upon the blood flow in the path of the light.
U.S. Pat. No. 4,945,896 teaches the use of a multiprobe sensor, using independent microelectrodes implanted inside the brain tissue, for measuring various parameters indicative of the function of the brain. This device includes a laser Doppler flow probe for measuring cerebral blood flow, and a probe for monitoring redox state (NADH). These probes can be mounted sequentially, i.e., one after another in the same housing, or they can be placed side by side. These devices suffer from a major drawback however. Tissue viability is not merely a reflection of various values of parameters measured at different times in one place, or different places at one time. The complex biochemical mechanisms that determine tissue viability are such that short time deviations between measurement at short distances between points of measurement can provide inaccurate or even misleading information. Thus, while the values of blood flow and redox state (NADH) must be monitored simultaneously at the same location, with the monitoring being for the same layer of tissue, this is not performed in the reference.
Another drawback encountered in NADH measurements is the Haemodynamic Artifact. This refers to an artifact in which NADH fluorescence measurements in-vivo are underestimated or overestimated due to the haemoglobin present in blood circulation, which absorbs radiation at the same wavelengths as NADH, and therefore interferes with the ability of the light to reach the NADH molecules. The haemoglobin also partially absorbs the NADH fluorescence. In particular, a reduction of haemoglobin in blood circulation causes an increase in fluorescence, generating a false indication of the true oxidation reduction state of the organ. U.S. Pat. No. 4,449,535 teaches, as means to compensate for this artifact, the monitoring of the concentration of red blood cells, by illuminating at a red wavelength (805 nm) simultaneously and in the same spot as the UV radiation required for NADH excitation and measuring the variation in intensity of the reflected red radiation, as well as the fluorescence at 440–480 nm, the former being representative of the intra-tissue concentration of red blood cells. Similarly Kobayashi et al (Kobayashi S., Nishiki K., Kaede K., Ogata E. J. Appl. Physiol. 31, 93–96, 1971) used ultraviolet (UV) illumination at 366 nm for NADH excitation, and red light at 720 nm for reflection measurements. However, U.S. Pat. No. 4,449,535 has at least two major drawbacks; firstly, and as acknowledged therein, using a single optical fiber to illuminate the organ, as well as to receive emissions therefrom causes interference between the outgoing and incoming signals, and certain solutions with different degrees of effectiveness are proposed. More importantly, though, two different wavelengths are used for illuminating the organ. FIG. 2 (based on Eggert & Blazek, 1987, © the Congress of Neurological Surgeons, Lippincott Williams & Wilkins) illustrates the penetration depth profile for various tissues of the human brain as a function of illuminating radiation wavelength, showing a plateau of relative insensitivity, of penetration depth (PD) with wavelength, for a wavelength range between about 360 nm and about 440 nm. For illuminating wavelengths greater than 440 nm, the penetration depth increases sharply with wavelength. Similar characteristics are found with other organs of the body. Thus, as may be seen from FIG. 2, the use of light radiation at the red end of the spectrum in accordance with U.S. Pat. No. 4,449,535 or as proposed by Kobayashi, to correct for blood haemodynamic artifacts in the NADH signal introduces inaccuracies into the measurements due to differences in penetration depths and therefore in the actual sampling volumes. Even though both radiation wavelengths are incident on the same spot, since detection is also at the same point, effectively two different elements of tissue, volume are being probed since the different radiation wavelengths penetrate the tissue to different depths. This results in measurements that are incompatible one with the other, the blood volume measurement relating to a greater depth of tissue than the NADH measurement. Therefore, the device disclosed by this reference does not enable adequate compensation of NADH to be effected using the simultaneous, though inappropriate, blood volume measurement. There is in fact no recognition of this problem, much less so any disclosure or suggestion of how to solve it. Further, there is no indication of how to measure other parameters such as blood flow rate or blood oxygenation level using the claimed apparatus.
In earlier patents; U.S. Pat. Nos. 5,916,171 and 5,685,313 (which have a common inventor with the present invention), a device is described that enables the monitoring of microcirculatory blood flow (MBF), the mitochondrial redox state (NADH fluorescence) and the microcirculatory blood volume (MBV), using a single source multi-detector electro-optical, fiber-optic probe device for monitoring various tissue characteristics to assess tissue vitality. During monitoring, the device is attached to the fore-mentioned tissue. The probe/tissue configuration enables front-face fluorometry/photometry. The two most important parameters involved in that fiber arrangement are the Optical Penetration Depth (PD) and the Averaged Sample Depth (SD), the PD parameter being dependent on both the tissue-type and on the irradiation wavelength; the SD parameter being dependent on the PD parameter and the distance between the ends of the excitation and collection fiber in contact with tissue.
Although U.S. Pat. No. 5,916,171 and U.S. Pat. No. 5,685,313 represent an improvement over the prior art, they nevertheless have some drawbacks:                (i)The oxidation level of the blood will introduce artifacts, affecting both the Mitochrondrial Redox State measurement (NADH fluorescence) and the microcirculatory blood volume (MBV) since these patents do not specify how to compensate for the oxygenation state of the blood in the tissue, i.e., the relative quantities of oxygenated blood to deoxygenated blood in the tissue. This problem is substantially overcome in the present invention by performing the NADH and blood volume measurements at an isosbestic point of the oxyhaemoglobin deoxyhaemoglobin absorption spectrum.        (ii)There is no facility included for measurement of the oxyhaemoglobin—deoxyhaemoglobin level, i.e. the Blood Oxygenation State, which is also an important tissue viability parameter, worthy of monitoring.        (iii) In these two U.S. patents, the same tissue volume needs to be monitored for all parameters, and the same light source and wavelength is used for the illumination needed for monitoring all three parameters. To measure both the NADH level and the blood flow rate, a relatively powerful UV laser is used. Using a relatively high intensity UV laser illumination source as proposed raises safety issues, especially for long-term monitoring.        (iv) The blood flow measurements impose several requirements on the UV laser source. In particular, the UV laser should have a high coherence length and very low intensity optical noise. Such lasers at these wavelengths are also not standard components and are indeed quite difficult to come by, which might lead to supply problems.        (v) There is no suggestion of monitoring Fp level, with or without any of the other parameters.        
It is an aim of the present invention to overcome the above deficiencies in the prior art.
Particularly, it is an aim of the present invention to provide a method and apparatus enabling the simultaneous in-vivo monitoring of blood flow rate (i.e. intravascular mean velocity times the number of moving red blood cells) and at least one, and preferably all, of the following: NADH concentration by fluorescence, total blood volume (i.e. concentration of red blood corpuscles) by reflectometry, blood haemoglobin oxygenation (i.e. the oxy/deoxy haemoglobin ratio) by fluorescence, flavoprotein concentration by fluorescence; for the same body tissue, in substantially the same layer within the same region. These parameters, which represent different biochemical and physiological activities of the tissue, are used to assess the tissue vitality in said layer and tissue region.
It is another aim of the invention to provide flexibility in design of apparatus for simultaneous measurement of four or five different parameters with reference to the same tissue layer.
It is another objective of the present invention to provide a method and apparatus for enabling the blood oxygenation of a tissue to be measured, which overcomes the deficiencies of the prior art.
It is another aim of the present invention to provide a method and apparatus for enabling the blood oxygenation of a tissue to be measured where prior art absorption methods cannot be used.
It is a further aim of this invention to enable the concurrent monitoring of blood parameters in different regions of the same organ.
It is a further aim of this invention to enable the concurrent monitoring of blood parameters in the same or different region of a number of different organs of the same type, for example the kidney of a number of patients.
It is a further aim of this invention to enable the concurrent monitoring of blood parameters in different organs belonging to the same or different patients.
Other objects and advantages of the invention will become apparent as the description proceeds.
These and other objectives are realised by the present invention by a revolutionary approach to tissue viability measurement, directed at a common tissue layer concept rather than based on necessarily using the same excitation wavelength for all parameters. The same tissue layer measurements can be achieved, as explained further on, by-utilizing several wavelengths that are all confined within a well defined wave-band, or alternatively by using even very different wavelengths and making adequate compensation for variable penetration depths, rather than being restricted to using a single radiation illumination as taught by U.S. Pat. No. 5,916,171 and U.S. Pat. No. 5,685,313.
Thus, the NADH fluorescence, blood volume and blood haemoglobin oxygenation state are measured using the same monochromatic illumination wavelength and the same detection fibers, ensuring that the same tissue volume is monitored for these three blood parameters. The illumination point is not coincident with the detection point, and the spacing between these points may be chosen according to the average sample depth that is desired.
The wavelength of the monochromatic light is chosen to lie at one of the isosbestic points of the extinction coefficient vs. wavelength curves for oxyhaemoglobin and deoxyhaemoglobin; wherein the NADH or blood volume measurements will be substantially unaffected by the oxygenation state of the blood.
The blood flow rate may be measured by Laser Doppler Flowmetry (LDF), typically using coherent light (a laser radiation), the illumination being applied at the same point on the tissue as for the above three parameters. Furthermore, this laser radiation can also be used for excitation of Fp fluorescence which enables the monitoring of flavoprotein concentration, which is an important physiological parameter, as discussed above.
However, the location of the detection fibers with respect to the illumination fibers, specifically the distance between their ends, is set to a different value to compensate for the different penetration depth of the two illuminating wavelengths, and thus to ensure that the same layer of tissue is monitored for the blood flow parameter and flavoprotein fluorescence, as is monitored for the other blood and tissue parameters. This distance will vary; both as a function of the tissue type being monitored and as a function of the selected wavelengths of the two illuminations. While it is generally preferable to monitor NADH and LDF over exactly the same tissue volume, not just layer, to achieve this aim, the excitation wavelengths used for the parameters being monitored should be confined within predefined wave-band for which penetration depth is substantially insensitive to wavelength.
Nonetheless, for any given type of tissue, there exists in general, a range of wavelengths with substantially the same penetration depth for each tissue type. For example, as illustrated in FIG. 2 for brain tissues, this plateau in penetration depth as a function of wavelength, extends from about 360 nm to about 440 nm, with some indication from other sources, that the plateau extends to even lower wavelengths. Similarly some other tissues feature similar plateaus at these or other wavelengths. If the monitored tissue is radiated using different wavelengths over the appropriate range, the penetration depth will be similar, and substantially the same volume of tissue may be monitored. In such cases, the same detection fibers may be used for both illuminating wavelengths.
Thus, the present invention also provides a method and apparatus for the measurement of blood oxygenation level based on fluorescence measurements, rather than reflection measurements. Essentially, a single radiation at a particular wavelength illuminates a tissue such as to stimulate the emission of fluorescence by the tissue. The intensity of the fluorescent radiation at two or more wavelengths (within the fluorescent radiation band) is measured, and the level of oxygenation is derived from these measurements. Such a method and apparatus is advantageously incorporated within the apparatus of the invention in which a number of tissue viability parameters are determined. Alternatively, a stand-alone device and corresponding method may be provided for the measurement of blood oxygenation level.
As discussed above, U.S. Pat. No. 5,916,171 and U.S. Pat. No. 5,685,313 are directed at the use of a single radiation at a single wavelength for monitoring a number of tissue viability parameters, including blood flow rate and NADH level. On the other hand, EP 442011 describes a sensor for non-invasive measurement of a single parameter, oxygen saturation in a tissue. In one embodiment, shown in FIG. 2 thereof, a carrier means has mounted therein a single light transmitter emitting electromagnetic waves of different wavelengths, and two receivers at different distances from the transmitter, each receiver being sensitive to a different one of these wavelengths reflected from the tissue.
Returning, to the references U.S. Pat. No. 5,916,171 and U.S. Pat. No. 5,685,313, a single optical fiber guide carries the single illuminating radiation to the tissue and receives light from the tissue via another fiber, and the received light is then directed into two separate channels. A single illuminating radiation is used to ensure that the same tissue volume is being considered for all the tissue vitality parameter measurements. Thus, not only would there be no motivation for a man of the art to consider these documents when desiring to provide an apparatus with two illuminating radiations at different wavelengths, these references actually teach against using more than one illuminating radiation source, and more so at different illuminating wavelengths. On the other hand, EP 422011 is directed exclusively at the measurement of a single parameter, oxygen saturation in a tissue, and does not consider in any shape or form the measurement of multiple tissue viability parameters such as blood flow rate and NADH—in fact it is not concerned with the measurement of two or more parameters, but rather uses both receivers to measure a single parameter. Thus, there would be no motivation for a man of the art to combine EP 442011 with U.S. Pat. No. 5,916,171 or U.S. Pat. No. 5,685,313 when seeking to provide a device according to the present invention. Moreover, even if the sensor of EP 442011 were to be combined with the apparatus of U.S. Pat. 5,916,171 or U.S. Pat. No. 5,685,313, the combination would not yield the present invention. For example, the apparatus of U.S. Pat. No. 5,916,171 and U.S. Pat. No. 5,685,313 does not provide radiation at a range of wavelengths, and the radiation is provided directly from a remote radiation source via optical fiber. This enables a relatively small tissue area to be monitored as the cross-section of the probe can therefore be quite small. On the other hand the sensor of EP 442011 has the transmitter itself (in the form of an LED) mounted onto the carrier, which therefore needs to be large enough to accommodate the same, and which has power leads, rather than optical fibers connecting the carrier to an external power source. Thus, these two devices—the apparatus and the sensor—are not compatible with each other, and very significant modifications to the two would be required to enable the sensor of EP 422011 to be incorporated into the apparatus of U.S. Pat. No. 5,916,171 and U.S. Pat. No. 5,685,313. This still leaves the question of how to configure the combination so that each of the receivers of EP 422011 is coupled to a different measuring channel of the apparatus of U.S. Pat. No. 5,916,171 and U.S. Pat. No. 5,685,313. More importantly, though, the sensor of EP 422011 is characterised in that the two receivers are mounted on the carrier in distances selected such that the lengths of the light paths through the tissue at the two different wavelengths are substantially equal. In such a case, by definition, the two different wavelengths must be directed to two different tissue layers, not to mention entirely different tissue volumes. Thus, not only would the fact that different tissue volumes are targeted by EP 422011 teach away from considering this reference in combination with U.S. Pat. No. 5,916,171 and U.S. Pat. No. 5,685,313 in the first place, such a combination still does not provide the apparatus of the present invention in which a single tissue layer is targeted by both radiations. In the present invention, the relative location of the detection fibers with respect to the illumination fibers, specifically the distance between their ends, is set to such as to compensate for the different penetration depths of the two illuminating wavelengths, and thus to ensure that the same layer of tissue is monitored for the blood flow parameter, as is monitored for the other blood and tissue parameters. Clearly, far from providing this arrangement, EP 422011 teaches away therefrom.
Regarding the determination of oxygenation level of a tissue according to the present invention, such a method and corresponding device are not disclosed or suggested in the prior art.
For example, WO 99/02956 uses a laser induced fluorescence method for assessing the levels of ischemia and hypoxia in a tissue, rather than blood oxygenation level. The method comprises the steps of (a) measuring the fluorescence spectra at two different points on the tissue; (b) calculating the tissue absorption spectrum from these measurements; and (c) calculating the intrinsic fluorescence spectrum. Thus, in order to perform the calculations for determining the absorption spectra, two different points on the tissue need to be considered, and thus the device requires two different detectors coupled to two corresponding measurement channels, in contrast to the present invention in which a single point on the tissue suffices for obtaining fluorescence measurements therefrom, which are in the form of intensity measurements. Further, there is no disclosure or suggestion of using the intensity of the fluorescent radiating at two or more wavelengths for determining ischemia or hypoxia, and less so for determining blood oxygenation level.
In U.S. Pat. No. 5,318,022 and in WO 98/44839, oximetry techniques are described, wherein in each case an excitation source of several wavelengths is used, and the intensity of the reflected radiation for each wavelength is measured, wherein the appropriate ratios of oxygen saturation are determined. In contrast, the present invention uses only a single wavelength, and the intensity of the fluorescent radiation emitted as a result thereof is measured at two or more fluorescent wavelengths, from which blood oxygenation level is determined.